Controlled differentiation of cells into specialized cell types depends on the detection of external and internal conditions and transduction of information to the transcription machinery to alter gene expression. In addition, normal growth and development requires the proper replication and segregation of chromosomes, and cells possess regulatory mechanisms that couple division or development to the fidelity of chromosomal transmission, Many diseases, including cancers, result from aberrant regulation of gene expression, and loss of fidelity of chromosome transmission. Microbial pathogenesis depends on the proper control of ene expression in response to changing environments. Our work focuses on the relatively simple developmental process of spore formation in Bacillus subtilis. Basic principles and mechanisms governing development, signal transduction, and gene expression are easily studied in B.subtilis by a combination of physiological, genetic, molecular, and biochemical approaches. Our work with B. subtilis will continue to contribute to a general understanding of mechanisms cells use to control growth, development, and gene expression. The long term goal of our research is to understand the signaling mechanisms that control the initiation of sporulation in B. subtilis. In particular, we are interested in the regulatory pathways controlling gene expression in response to developmental signals. External conditions important for the initiation of sporulation include nutrient deprivation and high cell density. In addition, for cells to begin to sporulate they must be replicating their DNA, have undamaged chromosomes, and have a functioning TCA cycle. All of these conditions control the activity of a multicomponent phospho-transfer pathway (the phosphorelay) that phosphorylates the transcription factor SpoOA. SpoOA-P then activates transcription of several genes needed for sporulation. The phosphorelay functions as a developmental checkpoint to ensure that sporulation begins only when conditions are appropriate. The primary focus of this proposal is to define the mechanisms and signaling pathways that control the phosphorelay in response to DNA replication and DNA damage. In addition, we will continue to dissect regulatory sites and trans-acting factors involved in gene expression early during development We will identify and characterize components of the signaling pathways in B. subtilis that control development in response to DNA replication and DNA damage by isolating mutations that make the initiation of sporulation and activation of SpoOA insensitive to signals related to DNA replication and DNA damage. Characterization of genes on the signaling pathways will provide insights into mechanisms cells use to sense whether their chromosomes are intact and replicated, and how gene expression and development are related accordingly.